Using T cell receptors (TCRs) as sensors, HIV-specific CD8+ T cells detect HIV peptides presented by human leukocyte antigen (pHLA) on antigen presenting cells (APCs) or target cells. The ability of HIV to readily mutate these peptides poses a great challenge for designing T cell-based vaccines and immunotherapies, since the escape variants are no longer effectively recognized by TCRs specific for the original peptide. How these molecular level mutations change their interactions with TCRs is not fully understood. Previous studies have focused on solution parameters, such as affinity, on-rate and off-rate, which were measured without external forces exerted on the interactions. These parameters, however, do not describe the real behavior of the pHLA-TCR interaction at the interface between T cells and APCs or target cells. T cells interact with APCs or target cells in a highly dynamic fashion, with constant membrane detachment and re-attachment. Therefore, the pHLA-TCR interaction is under constant mechanical stress. We believe that pHLA-TCR binding strength under mechanical force determines the specificity of pHLA-TCR interaction. Only bindings with sufficient strength allow the transfer of mechanical force originating from cell locomotion to TCR, and it is force-induced TCR conformational change that leads to TCR signaling and T cell response. Therefore, we hypothesize that HIV escape variants evade T cell attack by reducing the mechanical strength of their binding to specific TCRs. Here, taking advantage of recent advances in atomic force microscopy (AFM), we will test our hypothesis by measuring the mechanical strength of the bindings between HLA-A2 presenting HIV Gag peptide SL9 (HLA- A2-SL9) or its escape variants and SL9-specific 868 TCR, and then correlating this with the induced T cell response. In addition, we will examine the binding between HLA-A2-SL9 variants and a recently discovered 'supraphysiological'a11b6 TCR that recognizes SL9 and all its escape variants. We expect that the escape variants bind to the 868 TCR with less mechanical strength than wild type SL9, and that a11b6 TCR recognizes the escape variants by binding them with higher mechanical strength than wild type 868 TCR. Specifically, in Aim 1, using purified proteins of 868 TCR, a11b6 TCR, HLA-A2-SL9 and its variants, we will measure their binding strength under mechanical force with AFM. The forces it takes to rupture the binding (rupture force) will be correlated with their ability to induce T cell responses. In Aim 2, we will use AFM tips functionalized with HLA-A2-SL9 or its variants to directly probe T cells expressing 868 TCR or a11b6 TCR and measure rupture force. T cell signaling as indicated by intracellular calcium flux or PI3K activation will be monitored simultaneously using real time ratio imaging under a fluorescence microscope. Rupture force will be correlated with the level of T cell responses. By characterizing the interactions between TCR and HIV escape variants using AFM for the first time, our study will have direct implications for the design of vaccines and T cell-based immunotherapies for HIV infection. PUBLIC HEALTH RELEVANCE: HIV constantly mutates to escape from the immune system. To understand how the mutants evade immune detection, we employ a cutting edge biophysical tool, the atomic force microscopy, to dissect how the mutants change their interactions with the T cell receptor. Our data will help design effective HIV vaccines or immunotherapies for HIV infection.